The important biological roles that peptides and proteins play as hormones, enzyme inhibitors, substrates, and neurotransmitters has led to the use of peptides and/or peptide mimetics as therapeutic agents. The peptide's bioactive conformation, combining structural elements such as alpha-helices, beta-sheets, turns, and/or loops, is important as it allows for selective biological recognition of receptors, enzymes, and nucleic acids, thereby influencing cell-cell communication and/or controlling vital cellular functions, such as metabolism, immune defense, and cell division (Babine et al., Chem. Rev. (1997) 97:1359). Unfortunately, the utility of peptides as drugs is severely limited by several factors, including their rapid degradation by proteases under physiological conditions, their poor cell permeability, and their lack of binding specificity resulting from conformational flexibility.
The alpha-helix is one of the major structural components of peptides. However, alpha-helical peptides have a propensity for unraveling and forming random coils, which are, in most cases, biologically less active, or even inactive, and are highly susceptible to proteolytic degradation.
Many research groups have developed strategies for the design and synthesis of more robust peptides as therapeutics. For example, one strategy has been to incorporate more robust functionalities into the peptide chain while still maintaining the peptide's unique conformation and secondary structure (see, for example, Gante, Angew. Chem. Int. Ed. Engl. (1994) 33:1699-1720; Liskamp, Red. Trav. Chim. Pays-Bas (1994) 113:1; Giannis, Angew. Chem. Int. Ed. Engl. (1993) 32:1244; Bailey, Peptide Chemistry, Wiley, New York (1990), 182; and references cited therein). Another approach has been to stabilize the peptide via covalent cross-links (see, for example, Phelan et al., J. Am. Chem. Soc. (1997) 119:455; Leuc et al., Proc. Natl. Acad. Sci. USA (2003) 100: 11273; Bracken et al., J. Am. Chem. Soc. (1994) 116:6432; Yan et al., Bioorg. Med. Chem. (2004) 14:1403). However, the majority of reported approaches involved the use of polar and/or labile cross-linking groups.
“Peptide stapling” is a term coined for a synthetic methodology used to covalently join two olefin-containing side chains present in a polypeptide chain using an olefin metathesis reaction (J. Org. Chem. (2001) 66(16); Blackwell et al., Angew. Chem. Int. Ed. (1994) 37:3281). Stapling of a peptide using a hydrocarbon cross-linker created from an olefin metathesis reaction has bee shown to help maintain a peptide's native conformation, particularly under physiological conditions (U.S. Pat. No. 7,192,713; Schafineister et al., J. Am. Chem. Soc. (2000) 122:5891-5892; Walensky et al., Science (2004) 305:1466-1470; each of which is incorporated herein by reference). This strategy has been applied to the apoptosis-inducing BID-BH3 alpha-helix, resulting in a higher suppression of malignant growth of leukemia in an animal model compared to the unstapled peptide (Walensky et al., Science (2004) 305:1466-1470; U.S. Patent Application Publication No. 2005/02506890; U.S. Patent Application Publication No. 2006/0008848; each of which is incorporated herein by reference).